Affinity chromatography is commonly used for isolation and separation of proteins in solution. Wilchek et al., "Affinity Chromatography", Methods in Enzymology, volume 104, p. 3 (1984) describes conventional supports and activating agents used in affinity chromatography, as well as "newer and effective methods for the preparation and assay of affinity matrices." An adsorbent having a specific affinity for the protein to be isolated is used to biospecifically select the protein. The adsorbent is attached to a support. After the protein solution contacts and binds to the adsorbent, an eluent is added which removes the bound protein from the adsorbent. The resulting solution is highly purified separated protein.
Suitable adsorbents may have general affinity for certain classes of proteins, or specific affinity for proteins having binding sites for the immobilized adsorbent-ligand.
Zimmerman and Fulcher, U.S. Pat. No. Re. 32,011 (hereby incorporated by reference) describe purification of a plasma protein using monoclonal antibodies. Monoclonal antibodies having a specific affinity for a particular protein are prepared and covalently attached to a support such as glass beads, agarose and derivatives thereof. After binding the specific protein to the monoclonal antibody, a washing step removes the protein to produce a highly purified protein solution.
Although the product isolated by Zimmerman et al. is highly purified, there is often a loss of protein in the recovered preparation. The cause of the loss is not known, but one of the causes may be that the support used in protein purification interacts and binds to or internalizes the protein which is to be isolated, and is not removed from the support when eluent is applied to remove protein from the monoclonal antibody. Such interaction may be the result of direct reactivity between the support and protein. It is also thought that antibody which is to be externally bound to the support is instead internalized and rendered useless for isolating protein to which it is specific.
The present invention is a protein purification column comprising an organic substrate matrix having low reactivity to proteins. The matrix is capable of maintaining monoclonal antibodies attached thereto in an external configuration and preventing interaction with the protein to be bound to the antibody, a monoclonal antibody having a specific affinity for the protein to be isolated attached to the substrate, and means for linking the antibody to the substrate.
The present invention also is a method for isolating and purifying specific protein from a solution, wherein
1. Protein-specific monoclonal antibodies are attached to an organic substrate matrix such as that described above to form an antibody-substrate conjugate; and
2. Protein to be isolated, in an appropriate buffer solution, is contacted with the antibody-substrate conjugate.
An appropriate buffer may applied to remove non-antibody bound contaminants followed by an appropriate eluent to remove the protein from the monoclonal antibody.
Protein which fails to bind ("fall-through") to the antibody on initial contact (as described in step 2) can be reapplied after eluting the column.
The substrates may be, for example, fluoroplastic materials such as perfluorinated membrane, perfluoropolymer resin, and perfluorocarbon resin. Sperati, "Fluoroplastics", Modern plastics Encyclopedia 1986-1987, pages 22-25, hereby incorporated by reference, describes these materials in detail.
Perfluorinated membranes are based on perfluorinated ion exchange polymers that provide chemical and thermal stability similar to that of fluorocarbon resin. Attached to the fluoropolymer chains are perfluorinated cation exchange sites. The polymer is permeable to many cations and polar compounds, but impermeable to anions and non-polar species. The membranes are thin polymer films which are usually reinforced with a perfluorocarbon resin support cloth. They are useful as separators. In a typical application, a fluid containing one or more components is in contact with one side of the membrane, and the preferred component is transferred through the membrane under influence of a driving force such as concentration difference across the membrane, electric potential, or hydrostatic pressure. These membranes include sulfonic films, reinforced sulfonic films, and reinforced bimembranes comprised of carboxylic and sulfonic films. Waller and Van Scoyoc, "Catalysis with Nafion", Chemtech July 1987, pp. 438-441 describe use of perfluorinated membranes as catalyst support for superacid catalysis "for a wide variety of reactions in synthetic organic chemistry" and for "metal ion exchange."
Perfluorocarbon resins are polymers and copolymers formed from fluorocarbon materials such as tetrafluoroethylene and hexafluoropropylene. Because these materials are resistant to high temperatures and chemically inert to almost all industrial chemicals and solvents, they are widely used to provide corrosion protection in chemical processing equipment by lining pipes, valves, pumps, and vessels. Characteristics of this material are the very strong interatomic bonds between carbon and fluorine atoms, the highly effective shielding of the polymer's carbon backbone by fluorine atoms, and its high molecular weight compared to other polymers.
Perfluoropolymer resins are thermoplastic materials that are highly resistant to attack by chemicals and solvents that cause rapid deterioration of other plastics. These resins are less dense, tougher, stiffer, and exhibit higher tensile strength creep resistance than perfluorocarbon resins.
The art does not teach protein purification columns having substrates as described above. Furthermore, the art does not teach or suggest that monoclonal antibodies suitable for isolating specific proteins can be attached to these types of substrates as described above to provide nearly complete isolation and recovery of a specific protein from a solution containing various other materials.